AAC Accepts, published online ahead of print on 22 December 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.04374-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis
2
resistance to rifampicin
3
Yu-Tze Hornga, Wen-Yih Jengb, Yih-Yuan Chenc,d, Che-Hung Liua, Horng-Yunn Doud,
4
Jen-Jyh Leee, Kai-Chih Changa, Chih-Ching Chienf, Po-Chi Sooa*
5 6
a
7 8
Department of Laboratory Medicine and Biotechnology, Tzu Chi University, College of Medicine, 701 Section 3, Chung Yang Road, Hualien 970, Taiwan, R.O.C.
b
9
Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701 and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei 115, Taiwan,
10
R.O.C.
11
c
Department of Internal Medicine, Chiayi Christian Hospital, Chiayi, Taiwan
12
d
National Institute of Infectious Diseases and Vaccinology, National Health Research
13 14
Institutes, Zhunan, Miaoli, Taiwan, R.O.C. e
15 16 17
Department of Internal Medicine, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien 970, Taiwan, R.O.C.
f
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 320, Taiwan, R.O.C.
18 19
*Correspondence: Dr. Po-Chi Soo, e-mail: [email protected]
20
Present address: Department of Laboratory Medicine and Biotechnology, Tzu Chi
21
University, College of Medicine, 701 Section 3, Chung Yang Road, Hualien 970, Taiwan,
22
R.O.C.
23 24
Keywords: Mycobacterium tuberculosis; rifampicin resistant; rpoB gene
25 1
26
Abstract
27
Most rifampicin-resistant strains have been associated with mutations in an 81-bp
28
rifampicin resistance-determining region (RRDR) in the Mycobacterium tuberculosis gene
29
rpoB. However, when targeting this region alone, rifampicin-resistant strains with mutations
30
outside the RRDR would not be detected. In this study, among fifty-one rifampicin-resistant
31
clinical isolates analyzing by sequencing 1681-bp-long DNA fragments containing the RRDR,
32
47 isolates contained mutations within the RRDR, three isolates have mutation both within
33
and outside of RRDR while only one isolate had single missense mutation (Arg548His)
34
located outside the RRDR. The drug susceptibility test of recombinant Mycobacterium
35
smegmatis and M. tuberculosis carrying mutated rpoB (Arg548His) showed an increased
36
minimum inhibitory concentration (MIC) for rifampicin, compared to control strains.
37
Modelling the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain
38
formed a more stable hydrogen-bond structure than Arg548, reducing the flexibility of the
39
rifampicin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant
40
does not effectively interact with rifampicin and resulting in bacterial resistance to the drug.
41
This is the first report on the relationship between the mutation of codon 548 of RpoB and
42
rifampicin resistance in tuberculosis. The novel mutational profile of the rpoB gene described
43
here will contribute to the comprehensive understanding of rifampicin resistance patterns and
44
to the development of a useful tool for simple and rapid drug susceptibility tests. 2
45
Introduction
46
Tuberculosis (TB) is an infectious diseases caused by Mycobacterium tuberculosis.
47
Globally, an estimated 3.6% of new TB cases and 20.2% of previously treated TB cases are
48
considered multidrug-resistant TB (MDR-TB) (1). In Taiwan, MDR-TB comprised 0.9% of
49
new cases and 6.7% of previously treated cases of TB in 2011 (2). Rifampicin is a first-line
50
anti-TB drug that is often associated with treatment failure (3, 4). The bactericidal mechanism
51
of rifampicin functions by binding to the β subunit of RNA polymerase, the product of the
52
rpoB gene (5), and thus suppressing transcription in bacterial cells. More than 96% of
53
rifampicin-resistant strains have mutations within the 81-bp rifampicin resistance-determining
54
region (RRDR) of the rpoB gene (codons 507 to 533) (4). Additionally, the frequency of
55
codon mutations in rpoB of rifampicin-resistant M. tuberculosis isolates varies across different
56
geographical regions. The most common mutations in the RRDR are found in codons 526 and
57
531 and account for 62.5%~81.1% of rifampicin-resistant strains (6-8). However, not all
58
mutations within the RRDR lead to the same level of rifampicin resistance. Amino acid
59
alterations in codon 526 or 531 cause greater resistance in bacteria than do mutations in
60
codons 511, 516, 518, or 522 (9). Outside the RRDR, rare rifampicin-resistant mutations have
61
been reported in codons 176 (Val176Phe) (10), 381 (Ala381Val) (11), 490 (Gln490His) (12),
62
500 (Ala500Val), 502 (Ile502Val), 505 (Phe505Ser), 538 (Leu538Phe) (13), 146 (Val146Phe)
63
and 572 (Ile572Phe) (4, 14). Thus, reference laboratories using molecular methods to examine 3
64
only the 81-bp RRDR may miss strains in which rifampicin resistance is suspected but where
65
no mutation is found.
66
Molecular surveillance of rifampicin-resistant M. tuberculosis isolates in western,
67
northern and southern Taiwan has been reported in the past decade (6, 15-17). However,
68
similar studies in eastern Taiwan, which accounts for two-thirds of the country and is
69
characterised by rugged mountains, have not been carried out to a sufficient degree. The ethnic
70
groups and lifestyles of the people in eastern Taiwan, which account for approximately 4.4%
71
of the total population, are very different from those in other regions of the country. Here, we
72
studied the prevalence of rpoB mutations associated with rifampicin-resistant M. tuberculosis
73
isolates in eastern Taiwan. We found one novel rpoB allele and constructed recombinant M.
74
tuberculosis and Mycobacterium smegmatis strains carrying this mutated rpoB gene to
75
demonstrate that it plays a role in bacterial resistance to rifampicin.
76
4
77
MATERIALS AND METHODS
78
Bacterial strains, plasmids and media. The clinical M. tuberculosis isolates were collected
79
from Tzu Chi Hospital in Hualien, which is located in eastern Taiwan, from 2011-2012. The
80
isolation rate is 7.67%. Among these isolates, 51 were resistant to rifampicin. The other
81
laboratory strains and plasmids used in this study are listed in Table 1. M. smegmatis mc2155
82
and M. tuberculosis H37Rv were used as mycobacterial hosts to carry recombinant plasmids
83
for drug susceptibility testing. M. smegmatis mc2155, M. tuberculosis H37Rv and their
84
transformants were cultured in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA)
85
supplemented with 10% Tween 80 or on 7H11 agar supplemented with Oleic Albumin
86
Dextrose Catalase (OADC, Difco Laboratories, Detroit, MI, USA). Escherichia coli DH5α
87
was used for DNA cloning and was incubated at 37 ⁰C in Luria-Bertani (LB) medium.
88
Bacteria containing the pGEM-T easy vector (Promega, Wisconsin, USA) were grown in LB
89
medium supplemented with ampicillin (50 g/ml). The primers designed in this study are
90
listed in Table 2.
91 92
Specimen collection and processing. Sputum specimens were liquefied and decontaminated
93
by the standard N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method. The sediment
94
from each specimen was inoculated onto the culture media and used for acid-fast staining. The
95
bacterial isolates were identified by conventional methods that included routine microscopy, 5
96
culture and positive nitrate and niacin tests (18).
97 98
Mycobacterial DNA extraction, PCR amplification and DNA sequencing. The
99
mycobacterial chromosomal DNA grown on Middlebrook 7H11 agar plates was extracted
100
according to procedure described in the previous study (19). In brief, aliquot of the bacterial
101
pellet was lysed in lysis buffer (KOH, pH 13.1) at 95 ℃ for 15 min before being neutralized
102
by neutralization buffer (HCl and acetic acid, pH 1.2). Aliquot of crude extract suspension was
103
used in polymerase chain reactions (PCRs). The rpoB fragment were amplified by PCR in a
104
Biometra uno-thermoblock (Biometra, Goettingen, Germany) using the primer pair
105
rpoB-FP/rpoB-RP (Table 2). The PCR reactions began with five-minute denaturation at 95 °C,
106
followed by 30 cycles of denaturation at 95 °C for one minute, annealing at 57 °C for one
107
minute, extension at 72 °C for two minutes, and a final extension at 72 °C for two minutes.
108
Next, DNA sequencing of the PCR fragments was performed by Tri-I Biotech Inc. (New
109
Taipei City, Taiwan) using either rpoB-FP, rpoB-RP, rpoB-seq-F or rpoB-seq-R primer. The
110
rpoB-seq-F and rpoB-seq-R primers were designed for confirming the sequence of 693-bp
111
DNA region including RRDR. The sequences obtained from the 51 clinical isolates were
112
compared with the known sequence of M. tuberculosis H37Rv rpoB (the accession number is
113
NC_000962.3).
114 6
115
Construction of a recombinant Mycobacterial strain carrying exogenous rpoB.
116
Wild-type and mutated rpoB DNA fragments were amplified by PCR using the primer pair
117
rpoB-full-cF/rpoB-full-cR (Table 2), and chromosomal DNA from M. tuberculosis H37Rv, a
118
clinical M. tuberculosis isolate with the Ser531Leu mutation in rpoB and a clinical M.
119
tuberculosis isolate with the Arg548His mutation in rpoB (MTBR548H) were used as
120
templates. The PCR reactions began with a five-minute denaturation at 95 °C, followed by 30
121
cycles of denaturation at 95 °C for one minute, annealing at 58 °C for one minute, extension at
122
72 °C for two minutes, and a final step at 72 °C for two minutes. The PCR fragments were
123
cloned into the pGEM-T easy plasmid (Promega, Wisconsin, USA), followed by excision with
124
EcoR I/Hind III and ligation into pMV261 (Table 1) (20). The constructs were then confirmed
125
by DNA sequencing. To prepare M. tuberculosis and M. smegmatis competent cells, bacteria
126
were incubated in 7H9 broth containing 0.2 M glycine at 37 ⁰C with shaking at 220 rpm until
127
the OD600 reached 0.8~1.0. Subsequently, the bacterial cells were washed three times with
128
ice-cold 10% (w/v) glycerol. Finally, the competent cells were suspended in ice-cold 10%
129
(w/v) glycerol, and the recombinant plasmids pMV261::rpoB_WTc, pMV261::rpoB_S531Lc
130
and pMV261::rpoB_R548Hc were transformed respectively into competent M. tuberculosis
131
H37Rv and M. smegmatis mc2155 cells by electroporation (BTX ECM630 system, Harvard
132
Apparatus, MA, USA) at 2500 V, 1000 Ω and 25 μF.
133 7
134
Drug susceptibility testing. Drug susceptibility testing for mycobacterial clinical isolates was
135
determined by the standard agar proportion method using 1μg/mL of rifampicin as breakpoint
136
(21). The minimum inhibitory concentration (MIC) was determined using standard agar
137
proportion method on 7H11 agar for recombinant M. tuberculosis and broth microdilution
138
method for recombinant M. smegmatis (22). In brief, aliquots of recombinant M. smegmatis
139
(5×103 cells/ml) was inoculated into a 96-well microtitre plate containing 2-fold serial
140
dilutions of rifampicin (from 0.125 to 64 μg/mL) in Müller-Hinton broth with OADC. Then,
141
the bacteria were incubated at 37 ⁰C and 200 rpm for three to five days. Aliquots of
142
recombinant M. tuberculosis and 1:100 dilution control were inoculated on 7H11 agar
143
containing OADC and 2-fold serial dilutions of rifampicin (from 0.125 to 64 μg/mL). The
144
experiments of MIC test were performed three times. The MIC value for each strain was
145
defined as the lowest concentration of rifampicin needed to inhibit bacterial growth.
146 147
Structural modeling. Structural models of M. tuberculosis RpoB bound to rifampicin
148
or DNA were developed by SWISS-MODEL, a fully automated protein structure
149
homology-modelling server, using Protein Data Bank (PDB) accession code 1I6V (Thermus
150
aquaticus RNAP complexed with rifampicin) or 4G7Z (Thermus thermophiles RNAP
151
complexed with DNA) as the respective templates. The M. tuberculosis RpoB Arg548His
152
(using E. coli residue numbering) mutant was generated by Coot (23) using the RpoB-DNA 8
153
modelling structure. All structural models were optimised by energy minimisation using
154
REFMAC5 (24) in the CCP4 program suite (25, 26). Prior to generating structural figures, all
155
models were superimposed by the secondary structure matching (SSM) algorithm of the
156
PDBeFold server (27). The structural figures were produced using PyMOL (DeLano Scientific,
157
http://www.pymol.org).
158
9
159
RESULTS
160
Genetic analysis of rpoB genes in rifampicin-resistant M. tuberculosis isolated from
161
eastern Taiwan. Fifty-one rifampicin-resistant isolates were collected from eastern Taiwan.
162
To determine whether the DNA sequence outside of the RRDR of rpoB that codes for 507-533
163
of RpoB (using E. coli numbering) was associated with rifampicin resistance in M.
164
tuberculosis, a 1681-bp DNA fragment containing RRDR was amplified from each
165
rifampicin-resistant isolate by PCR and analysed by DNA sequencing. After comparison with
166
the rpoB sequence of M. tuberculosis H37Rv, the most commonly mutated sites in RpoB were
167
found to be codons 531 (74.5%) and 526 (21.6%), which are both located in the RRDR (Table
168
3). Of the 51 rifampicin-resistant isolates, 50 were mutated within the RRDR, while only one
169
clinical isolate, named MTBR548H, had a single mutation outside of this region. Although
170
there were three isolates having mutation, at codon 500, 552 and 576 respectively, outside of
171
RRDR, they had second or third mutation in the RRDR (Table 3). Most rifampicin-resistant
172
isolates (72.5%) had one mutated codon in RpoB; however, 23.5% and 3.9% of
173
rifampicin-resistant isolates had two and three mutated RpoB codons, respectively (Table 4).
174 175
Mutation of codon 548 in RpoB results in resistance to rifampicin in recombinant
176
M. tuberculosis and M. smegmatis. In the rifampicin-resistant isolates, one novel rpoB allele
177
that contained a single mutation, at codon 548, outside of the 81-bp RRDR was identified in 10
178
the clinical mutant, MTBR548H (Table 3). To determine whether the mutation of codon 548
179
(Arg548His) in RpoB was involved in bacterial resistance to rifampicin, we constructed
180
recombinant M. tuberculosis H37Rv and M. smegmatis mc2155 strains that carried mutated or
181
wild-type rpoB (Table 1). A bacterium carrying rpoB with the mutated codon Ser531Leu was
182
constructed as a positive control due to an association between this common missense
183
mutation of RpoB and high levels of mycobacterial rifampicin resistance (Table 1). Next, the
184
susceptibility of the recombinant strains to rifampicin was tested. The rifampicin MIC for the
185
recombinant M. smegmatis strain containing the wild-type M. tuberculosis rpoB gene (named
186
CH004) was 2 μg/ml. Meanwhile, the recombinant M. smegmatis strains containing RpoB
187
mutated at codon 531 or 548 (named CH006 and CH005, respectively) showed MIC values of
188
16 μg/mL and 4 μg/mL, respectively, which were elevated 8-fold and 2-fold compared with
189
strains containing either the vector control or wild-type rpoB (Table 5). Additionally, M.
190
tuberculosis H37Rv harbouring RpoB (Arg548His), named YY002, had an MIC of 2~4
191
μg/mL, which was elevated more than 16-fold compared with M. tuberculosis harbouring the
192
wild type rpoB (named YY003) (Table 6). The M. tuberculosis H37Rv harbouring RpoB with
193
mutated codon 531 (named YY004) was used as positive control showing rifampicin
194
resistance (Table 6). Thus, our results indicated that a missense mutation in RpoB at codon
195
548 (Arg548His), which is located outside of the RRDR, slightly increased the resistance of
196
mycobacteria to rifampicin. 11
197 198
Structural models of M. tuberculosis RpoB bound to rifampicin or promoter DNA.
199
To address the structural effect of the Arg548His RpoB mutation on M. tuberculosis
200
rifampicin resistance, structural models of RpoB bound to rifampicin or DNA were generated
201
by a homology-modelling server. In the RpoB-rifampicin complex model, it appeared that
202
residue Arg548 was shifted away from the rifampicin-binding pocket of RpoB and
203
nonessential residues for RpoB binding to rifampicin (Fig. 1). However, in the RpoB-DNA
204
complex model, it appeared that residue Arg548 was close to the rifampicin-resistant cluster II
205
region of RpoB (Fig. 1) (5). In the Arg548His mutant of the RpoB-DNA complex model, the
206
imidazole ring of His548 seemed to provide two hydrogen bonds, between His548 and Ile569
207
as well as between His548 and Ala543 (Fig. 1B), leading to increased rigidity of the
208
rifampicin-resistant cluster II region of RpoB (Fig. 1C). We also found dramatic variations in
209
the side-chain orientation of the rifampicin-resistant cluster II region of RpoB in the
210
rifampicin- and DNA-binding models (Fig. 1C). We speculated that the side chain of His548
211
could provide a more stable hydrogen bond-linking structure than Arg548, thus reducing the
212
flexibility of the rifampicin-resistant cluster II region of RpoB. Thus, the rifampicin-resistant
213
cluster II region of the Arg548His RpoB mutant was unable to interact effectively with
214
rifampicin, resulting in bacterial rifampicin resistance.
215
DISCUSSION 12
216
Although mycobacterial resistance to rifampicin is primarily mediated by mutations within the
217
RRDR, many studies have reported novel mutations outside this region, such as at codons 176
218
(Val176Phe) (10), 381 (Ala381Val) (11), 490 (Gln490His) (12), 500 (Ala500Val), 502
219
(Ile502Val), 505 (Phe505Ser), 538 (Leu538Pro) (13), 146 (Val146Phe) and 572 (Ile572Phe)
220
(14). These mutations were discovered using DNA sequencing of clinical rifampicin-resistant
221
M. tuberculosis isolates; however, most of these findings lacked in vivo confirmation of the
222
relationship between the mutated codon and drug resistance. An exception to this is the work
223
by Siu et al., who constructed recombinant M. smegmatis and M. tuberculosis to show that
224
mutations in codons 146 (Val146Phe) and 572 (Val572Phe) led to rifampicin resistance (14).
225
Compared with western, northern and southern Taiwan, the topography and population
226
composition are different in eastern Taiwan. Therefore, we screened the rpoB gene in 51
227
clinical rifampicin-resistant M. tuberculosis isolates in eastern Taiwan and identified one
228
isolate, MTBR548H, having a mutation in codon 548, which is located outside the RRDR and
229
had not been previously reported, as a cause of bacterial rifampicin resistance. To validate the
230
association of this mutated genotype to the rifampicin-resistant phenotype in MTBR548H, the
231
mutated rpoB of MTBR548H was cloned in multicopy-number plasmid and transformed into
232
wild-type M. tuberculosis and M. smegmatis to produce resistant mutant strains, YY002 and
233
CH005 respectively. The rifampicin susceptibilities of recombinant strains indicated that the
234
Arg548His mutation in RpoB contributed to rifampicin resistance in M. tuberculosis. This 13
235
finding was supported by data obtained from RpoB-DNA structural modelling, which showed
236
that RpoB residue His548 reduced the interaction between RpoB and rifampicin in the
237
RpoB-DNA complex. Our findings could help to improve the detection of mycobacterial
238
rifampicin resistance, and we suggest that position 548 should be included when the RRDR is
239
analysed by DNA sequencing due its close proximity to the RRDR. This can be readily
240
performed without increasing the turnaround time or reaction cost of single-sequencing
241
reactions.
242
Here, the most common mutations involved in rifampicin resistance were missense
243
mutations in codon 531 of rpoB (Table 3). These results were comparable to those of previous
244
studies (6), but the frequency of this mutation (74.5%) was much higher than in earlier reports
245
from other regions of Taiwan, including southern Taiwan (41.5% during 1996-1998), the
246
entire Taiwan (49.4% during 1998-2003) and central and northern Taiwan (68.2% during
247
1999-2011) (6, 15, 16). This difference in frequency could be due to the increasing prevalence
248
of rifampicin-resistant M. tuberculosis clones spreading through eastern Taiwan in recent
249
years (2011-2012).
250
In the recent study in Canada, only 2.9% of clinical rifampicin-resistant isolates (1/35)
251
had double point mutations in rpoB (28). However, we found high ratios of clinical
252
rifampicin-resistant isolates having two or three point mutations in rpoB. (23.5% and 3.9%
253
respectively, Table 4). In our study, most of double or triple point mutations in rpoB were still 14
254
located in RRDR (Table 3). The relationship between the multi-mutations in rpoB and MIC
255
values are not clear and needed further study.
256
The population of aboriginal people in eastern Taiwan is more than other regions in
257
Taiwan. In recent study, 5.43% of drug-resistant TB cases were rifampicin monoresistant in
258
aboriginal people in eastern Taiwan while 2.27% in aboriginal people in southern Taiwan (29).
259
In non-aboriginal people in southern Taiwan from 2010 to 2011 and eastern Taiwan from
260
2005 to 2008 as well as in people in Canada during 2012, no rifampicin-monoresistant clinical
261
isolate was found (30, 31). The novel mutant rpoB (R548H) found in our study might occur
262
due to this special populational and geographical environment. Such mutant strain may spread
263
to other regions in the future if the efforts of control of tuberculosis were not effective.
264
The mutation at codon 531 was associated with high-level resistance to rifampicin in
265
most clinical and recombinant strains (Table 3, 5 and 6) (15, 20). Extrapolation from the
266
crystal structure of the rifampicin-RNAP (RNA polymerase) complex showed that Ser531 in
267
the RpoB rifampicin-binding pocket formed a direct hydrogen bond with the critical hydroxyl
268
of rifampicin, O2. Thus, mutation of codon 531 reduced the interaction between rifampicin
269
and RpoB, resulting in bacterial rifampicin resistance (5). Siu et al. reported that mutation of
270
residues Val146 and Ile572, which are outside the RRDR, resulted in M. tuberculosis
271
resistance to rifampicin. In a model of rifapentine-RRDR (rather than rifampicin-RRDR),
272
residue 572 was localised to the wall of the rifampicin-binding pocket of RpoB, while residue 15
273
146 was located beneath the rifampicin-binding pocket and did not directly interact with the
274
drug. Mutation of residue 572 (Ile572Phe) likely reduced the affinity between that residue and
275
rifampicin, while mutation of residue 146 (Val146Phe) likely affected the folding and packing
276
of the rifampicin-binding pocket (14). In our study, RpoB residue Arg548, which is outside
277
the RRDR, was not located in the rifampicin-binding pocket in the RpoB-rifampicin model.
278
Therefore, residue Arg548 does not directly interact with rifampicin. However, a point
279
mutation (CGC to CAC) in codon 548, which changes the residue from Arg to His, led to
280
reduced flexibility in the RpoB structure (Fig. 1B and 1C). These results indicated that
281
rifampicin could not block the transcriptional activity of RNAP containing a mutated form of
282
RpoB when the RNAP was bound to DNA. Together, our results provide a potential
283
mechanism for the differences in rifampicin resistance between the RpoB-DNA and
284
RpoB-rifampicin models.
285 286
16
287
ACKNOWLEDGMENTS
288
This work was supported partly by grants (contract number NSC 101-2320 -B-320 -002 and
289
MOST 103-2320-B-320-008-MY3) from Ministry of Science and Technology and partly by
290
grants TCIRP99002-04 from Tzu Chi University. We thank the Core Facilities for Protein
291
Structural Analysis in Academia Sinica (Taipei, Taiwan) to assist us in structural modelling
292
and expanding. We also thank Mr. Chi-Hsien Fu and Miss Ying-Huei Chen in Department of
293
Laboratory Medicine Buddhist Tzu Chi General Hospital for their kind help to collect the
294
clinical mycobacterial isolates.
295
17
296
REFERENCES
297
1.
298 299
World Health Organization. 2013. Global tuberculosis report 2013. http://www.who.int/tb/publications/global_report/gtbr13_main_text.pdf.
2.
Centers for Disease Control DoH, R.O.C. (Taiwan). 2012. Taiwan tuberculosis control
300
report 2012
301 302
(http://www.cdc.gov.tw/infectionreportinfo.aspx?treeid=075874dc882a5bfd&nowtre eid=54c73190d38c0d49&tid=3AA3338E6C212AA3).
303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Mitchison DA, Nunn AJ. 1986. Influence of initial drug resistance on the response to short-course chemotherapy of pulmonary tuberculosis. The American review of respiratory disease 133:423-430. Somoskovi A, Parsons LM, Salfinger M. 2001. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respiratory research 2:164-168. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst SA. 2001. Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell 104:901-912. Jou R, Chen HY, Chiang CY, Yu MC, Su IJ. 2005. Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates and identification of 11 novel rpoB alleles in Taiwan. Journal of clinical microbiology 43:1390-1394. Ramirez MV, Cowart KC, Campbell PJ, Morlock GP, Sikes D, Winchell JM, Posey JE. 2010. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis. Journal of clinical microbiology 48:4003-4009. Kumar P, Balooni V, Sharma BK, Kapil V, Sachdeva KS, Singh S. 2014. High degree of multi-drug resistance and hetero-resistance in pulmonary TB patients from Punjab state of India. Tuberculosis 94:73-80. Ohno H, Koga H, Kohno S, Tashiro T, Hara K. 1996. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrobial agents and chemotherapy 40:1053-1056. Heep M, Rieger U, Beck D, Lehn N. 2000. Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy 44:1075-1077. Taniguchi H, Aramaki H, Nikaido Y, Mizuguchi Y, Nakamura M, Koga T, Yoshida S. 1996. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol Lett 144:103-108. Minh NN, Van Bac N, Son NT, Lien VT, Ha CH, Cuong NH, Mai CT, Le TH. 2012. Molecular characteristics of rifampin- and isoniazid-resistant Mycobacterium 18
333 334
tuberculosis strains isolated in Vietnam. Journal of clinical microbiology 50:598-601. 13.
Yue J, Shi W, Xie J, Li Y, Zeng E, Wang H. 2003. Mutations in the rpoB gene of
335
multidrug-resistant Mycobacterium tuberculosis isolates from China. Journal of clinical
336
microbiology 41:2209-2212.
337
14.
Siu GK, Zhang Y, Lau TC, Lau RW, Ho PL, Yew WW, Tsui SK, Cheng VC, Yuen KY, Yam
338
WC. 2011. Mutations outside the rifampicin resistance-determining region associated
339 340
with rifampicin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 66:730-733.
341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
Hwang HY, Chang CY, Chang LL, Chang SF, Chang YH, Chen YJ. 2003. Characterization of rifampicin-resistant Mycobacterium tuberculosis in Taiwan. Journal of medical microbiology 52:239-245. Lin YH, Tai CH, Li CR, Lin CF, Shi ZY. 2013. Resistance profiles and rpoB gene mutations of Mycobacterium tuberculosis isolates in Taiwan. J Microbiol Immunol Infect 46:266-270. Tseng ST, Tai CH, Li CR, Lin CF, Shi ZY. 2013. The mutations of katG and inhA genes of isoniazid-resistant Mycobacterium tuberculosis isolates in Taiwan. J Microbiol Immunol Infect. Nolte FS, Metchock B, Williams T, Diem L, Bressler A, Tenover FC. 1995. Detection of penicillin-resistant Streptococcus pneumoniae with commercially available broth microdilution panels. Journal of clinical microbiology 33:1804-1806. Soo PC, Horng YT, Chang KC, Wang JY, Hsueh PR, Chuang CY, Lu CC, Lai HC. 2009. A simple gold nanoparticle probes assay for identification of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex from clinical specimens. Molecular and cellular probes 23:240-246. Nakata N, Kai M, Makino M. 2012. Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis. Antimicrobial agents and chemotherapy 56:2008-2013. Clinical and Laboratory Standards Institute. 2011. Susceptibility Testing of Mycobacteria, Nocardia and Other Aerobic Actinomycetes: Approved Standard-second edition M24-A2. CLSI, Wayne, PA, USA. Wiegand I, Hilpert K, Hancock RE. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163-175. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta crystallographica. Section D, Biological crystallography 60:2126-2132. Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta crystallographica. Section D, Biological crystallography 67:355-367. 19
371
25.
Collaborative Computational Project N. 1994. The CCP4 suite: programs for protein
372
crystallography. Acta crystallographica. Section D, Biological crystallography
373
50:760-763.
374
26.
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM,
375
Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS,
376
Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS. 2011. Overview of the CCP4
377 378
suite and current developments. Acta crystallographica. Section D, Biological crystallography 67:235-242.
379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398
27.
28.
29.
30. 31.
32. 33.
Krissinel E, Henrick K. 2004. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta crystallographica. Section D, Biological crystallography 60:2256-2268. Jamieson FB, Guthrie JL, Neemuchwala A, Lastovetska O, Melano RG, Mehaffy C. 2014. Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis. Journal of clinical microbiology 52:2157-2162. Chen YY, Chang JR, Huang WF, Kuo SC, Yeh JJ, Lee JJ, Jang CS, Sun JR, Chiueh TS, Su IJ, Dou HY. 2014. Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006-2011. The Journal of infection 68:332-337. Public Health Agency of Canada. 2012. Tuberculosis: Drug resistance in Canada-2012. http://www.phac-aspc.gc.ca/tbpc-latb/pubs/tb-dr2012/index-eng.php#results_1. Chen YY, Tseng FC, Chang JR, Kuo SC, Lee JJ, Yeh JJ, Chiueh TS, Sun JR, Su IJ, Dou HY. 2014. Distinct modes of transmission of tuberculosis in aboriginal and non-aboriginal populations in taiwan. PloS one 9:e112633. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. Journal of molecular biology 166:557-580. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, Bansal GP, Young JF, Lee MH, Hatfull GF, et al. 1991. New use of BCG for recombinant vaccines. Nature 351:456-460.
399
20
400
Figure legend
401
Figure 1. Structural models of M. tuberculosis RpoB that bound with rifampicin or promoter
402
DNA. (A) Superimposition of the models of M. tuberculosis RpoB that bound with rifampicin
403
(protein in gray cartoon model) or promoter DNA (protein in light-orange cartoon model). The
404
rifampicin is shown in stick model (carbon atoms in dark-green). The DNA is shown in sphere
405
model (carbon atoms in light-green and yellow-orange). The carbon atoms of key residues for
406
the RpoB-rifampicin complex, RpoB-DNA complex, and RpoB mutant-DNA complex are
407
shown in light-green, cyan, magenta line models, respectively. Oxygen atoms are shown in red,
408
nitrogen atoms are in blue, and phosphorus atoms are in orange. (B) and (C) Close-up view of
409
the Arg548 located loop and the rifampicin-resistant cluster II region of RpoB. The potential
410
hydrogen bonds are indicated by black dashed lines. The color indication of residues numbers
411
are the same as the color painting of line models. The residues number are labeled according
412
to the E. coli RpoB protein sequence. Values in the parentheses are for M. tuberculosis RpoB
413
protein sequence. (D) Protein sequence alignment of the Arg548 located loop and the
414
rifampicin-resistant cluster II loop of four prokaryotic RpoB. The rifampicin-resistant cluster
415
II region of RpoB is marked with black bar. The Arg548His mutant site and the residues
416
interacting with His548 by potential hydrogen-bond are indicated by black asterisk and dots,
417
respectively.
418
21
419
Table 1. Strains and plasmids used in this study. Strain or plasmid
descriptions
Reference or source
Clinical isolates resistant to rifampicin not to isoniazid
This study
E. coli DH5α
supE44∆lacU169 (f80 lacZ∆M15)hsdR1 recA1 endA1 gyrA96 thi-1 relA1
(32)
M. smegmatis mc2155
Wild type
ATCC700084
Strains M. tuberculosis MTBR548H
2
CH003
M. smegmatis mc 155 / pMV261 vector only, Kmr
This study
CH004
M. smegmatis mc2155 / pMV261::rpoB_WTc , Kmr
This study
CH005
M. smegmatis mc2155/ pMV261::rpoB_R548Hc, Kmr
This study
CH006
M. smegmatis mc2155/ pMV261::rpoB_S531Lc, Kmr
This study
Wild type
ATCC27294
YY001
M. tuberculosis H37Rv / pMV261 vector only, Kmr
This study
YY002
M. tuberculosis H37Rv / pMV261::rpoB_R548Hc, Kmr
This study
YY003
M. tuberculosis H37Rv / pMV261::rpoB_WTc, Kmr
This study
YY004
M. tuberculosis H37Rv / pMV261::rpoB_S531Lc, Kmr
This study
pGEM-T easy
Cloning vector, Ampr
Promega, Wisconsin, USA
pMV261
Mycobacterium species/E. coli shuttle vector, Kmr pMV261 containing wild-type rpoB from M. tuberculosis H37Rv, Kmr
(33)
pMV261::rpoB_R548Hc
pMV261 containing mutated rpoB from rifampicin-resistant M. tuberculosis, codon 548 of RpoB was changed to histidine Kmr
This study
pMV261::rpoB_S531Lc
pMV261 containing mutated rpoB from rifampicin-resistant M. tuberculosis, codon 531 of RpoB was changed to lysine, Kmr
This study
M. tuberculosis H37Rv
plasmids
pMV261::rpoB_WTc
420 22
This study
421
Table 2 primers designed in this study Primer
Sequence (5’-3’)
Amplicon size
rpoB-FP
CCCGCCAGAGCAAAACAGC
rpoB-RP
TACTCCACCTCGCCCGCC
rpoB-seq-F
AATATCTGGTCCGCTTGCAC
rpoB-seq-R
ACGAGGGCACGTACTCCA
rpoB-full-cF
GCGAATTCGGAAGGAAAAATGGCAGATTCCCGCCAG
rpoB-full-cR
GCAAGCTTTTACGCAAGATCCTCGACAC
1681 bp
422
23
3545 bp
423
Table3. Mutation in rpoB gene in 51 rifampicin-resistant M. tuberculosis Mutated codon Ala500Gly
AGCAAC
1
2.0
a
ATGTTG
1
2.0
a
GACGTC
3
5.9
a
CACTAC
1
2.0
a
His526Asn
Arg548His Pro552Ser Pro567Ser
424 425 426 427 428 429 430
Frequency (%)
1
Asp516Val
Leu533Pro
b
GCCGGC
Met515Leu
Ser531Leu
Number of strains
a
Ser512Asn
His526Tyr
Nucleic acid change
2.0
CACAAC
10
19.6
a
TCGTTG
38
74.5
a
CTGCCG
10
19.6
1
2.0
CGCCAC
e
CCGTCG CCTTCT
1
c
1
2.0
d
2.0
Note: a, The mutations located within the 81-bp RRDR of rpoB gene. There were 11 isolates having two mutated codons both in the RRDR b, The isolate had three mutated codons in RpoB: Ala500Gly, Met515Leu and Asp516Val. c, The isolate had two mutated codons in RpoB: Ser531Leu and Pro552Ser. d, The isolate had three mutated codons in RpoB: Ser531Leu, Leu533Pro and Pro567Ser. e, The novel allele was identified in this study.
431
24
432
Table 4. The frequency of single or multi mutations in RpoB in 51
433
rifampicin-resistant M. tuberculosis Mutation in RpoB
No. of strains
Frequency
One codon
37
72.5%
Two codons
12
23.5%
Three codons
2
3.9%
Four codons
0
0%
434
25
435 436
Table 5. Rifampicin susceptibility of the recombinant M. smegmatis strains with wild type RpoB or mutated RpoB M. smegmatis strains
Codon no. and amino acid
Rifampicinb MIC(μg/mL)
CH003
pMV261a
2
CH004
wild type rpoB
2
CH005
Arg548His
4
CH006
Ser531Leu
16
437
a, Vector control
438
b, Each MIC test was performed three times.
439
26
440 441
Table 6. Rifampicin susceptibility of the recombinant M. tuberculosis strains with wild type RpoB or mutated RpoB using agar proportion method . 10-4 dilutionb
10-2 dilutionb M. tuberculosis strains
Codon no. and amino acid
Rifampicinc MIC(μg/mL)
Sensitive /Resistantd
Rifampicinc MIC(μg/mL)
Sensitive /Resistantd
YY001
pMV261 a
1
Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis
2
resistance to rifampicin
3
Yu-Tze Hornga, Wen-Yih Jengb, Yih-Yuan Chenc,d, Che-Hung Liua, Horng-Yunn Doud,
4
Jen-Jyh Leee, Kai-Chih Changa, Chih-Ching Chienf, Po-Chi Sooa*
5 6
a
7 8
Department of Laboratory Medicine and Biotechnology, Tzu Chi University, College of Medicine, 701 Section 3, Chung Yang Road, Hualien 970, Taiwan, R.O.C.
b
9
Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701 and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei 115, Taiwan,
10
R.O.C.
11
c
Department of Internal Medicine, Chiayi Christian Hospital, Chiayi, Taiwan
12
d
National Institute of Infectious Diseases and Vaccinology, National Health Research
13 14
Institutes, Zhunan, Miaoli, Taiwan, R.O.C. e
15 16 17
Department of Internal Medicine, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien 970, Taiwan, R.O.C.
f
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 320, Taiwan, R.O.C.
18 19
*Correspondence: Dr. Po-Chi Soo, e-mail: [email protected]
20
Present address: Department of Laboratory Medicine and Biotechnology, Tzu Chi
21
University, College of Medicine, 701 Section 3, Chung Yang Road, Hualien 970, Taiwan,
22
R.O.C.
23 24
Keywords: Mycobacterium tuberculosis; rifampicin resistant; rpoB gene
25 1
26
Abstract
27
Most rifampicin-resistant strains have been associated with mutations in an 81-bp
28
rifampicin resistance-determining region (RRDR) in the Mycobacterium tuberculosis gene
29
rpoB. However, when targeting this region alone, rifampicin-resistant strains with mutations
30
outside the RRDR would not be detected. In this study, among fifty-one rifampicin-resistant
31
clinical isolates analyzing by sequencing 1681-bp-long DNA fragments containing the RRDR,
32
47 isolates contained mutations within the RRDR, three isolates have mutation both within
33
and outside of RRDR while only one isolate had single missense mutation (Arg548His)
34
located outside the RRDR. The drug susceptibility test of recombinant Mycobacterium
35
smegmatis and M. tuberculosis carrying mutated rpoB (Arg548His) showed an increased
36
minimum inhibitory concentration (MIC) for rifampicin, compared to control strains.
37
Modelling the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain
38
formed a more stable hydrogen-bond structure than Arg548, reducing the flexibility of the
39
rifampicin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant
40
does not effectively interact with rifampicin and resulting in bacterial resistance to the drug.
41
This is the first report on the relationship between the mutation of codon 548 of RpoB and
42
rifampicin resistance in tuberculosis. The novel mutational profile of the rpoB gene described
43
here will contribute to the comprehensive understanding of rifampicin resistance patterns and
44
to the development of a useful tool for simple and rapid drug susceptibility tests. 2
45
Introduction
46
Tuberculosis (TB) is an infectious diseases caused by Mycobacterium tuberculosis.
47
Globally, an estimated 3.6% of new TB cases and 20.2% of previously treated TB cases are
48
considered multidrug-resistant TB (MDR-TB) (1). In Taiwan, MDR-TB comprised 0.9% of
49
new cases and 6.7% of previously treated cases of TB in 2011 (2). Rifampicin is a first-line
50
anti-TB drug that is often associated with treatment failure (3, 4). The bactericidal mechanism
51
of rifampicin functions by binding to the β subunit of RNA polymerase, the product of the
52
rpoB gene (5), and thus suppressing transcription in bacterial cells. More than 96% of
53
rifampicin-resistant strains have mutations within the 81-bp rifampicin resistance-determining
54
region (RRDR) of the rpoB gene (codons 507 to 533) (4). Additionally, the frequency of
55
codon mutations in rpoB of rifampicin-resistant M. tuberculosis isolates varies across different
56
geographical regions. The most common mutations in the RRDR are found in codons 526 and
57
531 and account for 62.5%~81.1% of rifampicin-resistant strains (6-8). However, not all
58
mutations within the RRDR lead to the same level of rifampicin resistance. Amino acid
59
alterations in codon 526 or 531 cause greater resistance in bacteria than do mutations in
60
codons 511, 516, 518, or 522 (9). Outside the RRDR, rare rifampicin-resistant mutations have
61
been reported in codons 176 (Val176Phe) (10), 381 (Ala381Val) (11), 490 (Gln490His) (12),
62
500 (Ala500Val), 502 (Ile502Val), 505 (Phe505Ser), 538 (Leu538Phe) (13), 146 (Val146Phe)
63
and 572 (Ile572Phe) (4, 14). Thus, reference laboratories using molecular methods to examine 3
64
only the 81-bp RRDR may miss strains in which rifampicin resistance is suspected but where
65
no mutation is found.
66
Molecular surveillance of rifampicin-resistant M. tuberculosis isolates in western,
67
northern and southern Taiwan has been reported in the past decade (6, 15-17). However,
68
similar studies in eastern Taiwan, which accounts for two-thirds of the country and is
69
characterised by rugged mountains, have not been carried out to a sufficient degree. The ethnic
70
groups and lifestyles of the people in eastern Taiwan, which account for approximately 4.4%
71
of the total population, are very different from those in other regions of the country. Here, we
72
studied the prevalence of rpoB mutations associated with rifampicin-resistant M. tuberculosis
73
isolates in eastern Taiwan. We found one novel rpoB allele and constructed recombinant M.
74
tuberculosis and Mycobacterium smegmatis strains carrying this mutated rpoB gene to
75
demonstrate that it plays a role in bacterial resistance to rifampicin.
76
4
77
MATERIALS AND METHODS
78
Bacterial strains, plasmids and media. The clinical M. tuberculosis isolates were collected
79
from Tzu Chi Hospital in Hualien, which is located in eastern Taiwan, from 2011-2012. The
80
isolation rate is 7.67%. Among these isolates, 51 were resistant to rifampicin. The other
81
laboratory strains and plasmids used in this study are listed in Table 1. M. smegmatis mc2155
82
and M. tuberculosis H37Rv were used as mycobacterial hosts to carry recombinant plasmids
83
for drug susceptibility testing. M. smegmatis mc2155, M. tuberculosis H37Rv and their
84
transformants were cultured in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA)
85
supplemented with 10% Tween 80 or on 7H11 agar supplemented with Oleic Albumin
86
Dextrose Catalase (OADC, Difco Laboratories, Detroit, MI, USA). Escherichia coli DH5α
87
was used for DNA cloning and was incubated at 37 ⁰C in Luria-Bertani (LB) medium.
88
Bacteria containing the pGEM-T easy vector (Promega, Wisconsin, USA) were grown in LB
89
medium supplemented with ampicillin (50 g/ml). The primers designed in this study are
90
listed in Table 2.
91 92
Specimen collection and processing. Sputum specimens were liquefied and decontaminated
93
by the standard N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method. The sediment
94
from each specimen was inoculated onto the culture media and used for acid-fast staining. The
95
bacterial isolates were identified by conventional methods that included routine microscopy, 5
96
culture and positive nitrate and niacin tests (18).
97 98
Mycobacterial DNA extraction, PCR amplification and DNA sequencing. The
99
mycobacterial chromosomal DNA grown on Middlebrook 7H11 agar plates was extracted
100
according to procedure described in the previous study (19). In brief, aliquot of the bacterial
101
pellet was lysed in lysis buffer (KOH, pH 13.1) at 95 ℃ for 15 min before being neutralized
102
by neutralization buffer (HCl and acetic acid, pH 1.2). Aliquot of crude extract suspension was
103
used in polymerase chain reactions (PCRs). The rpoB fragment were amplified by PCR in a
104
Biometra uno-thermoblock (Biometra, Goettingen, Germany) using the primer pair
105
rpoB-FP/rpoB-RP (Table 2). The PCR reactions began with five-minute denaturation at 95 °C,
106
followed by 30 cycles of denaturation at 95 °C for one minute, annealing at 57 °C for one
107
minute, extension at 72 °C for two minutes, and a final extension at 72 °C for two minutes.
108
Next, DNA sequencing of the PCR fragments was performed by Tri-I Biotech Inc. (New
109
Taipei City, Taiwan) using either rpoB-FP, rpoB-RP, rpoB-seq-F or rpoB-seq-R primer. The
110
rpoB-seq-F and rpoB-seq-R primers were designed for confirming the sequence of 693-bp
111
DNA region including RRDR. The sequences obtained from the 51 clinical isolates were
112
compared with the known sequence of M. tuberculosis H37Rv rpoB (the accession number is
113
NC_000962.3).
114 6
115
Construction of a recombinant Mycobacterial strain carrying exogenous rpoB.
116
Wild-type and mutated rpoB DNA fragments were amplified by PCR using the primer pair
117
rpoB-full-cF/rpoB-full-cR (Table 2), and chromosomal DNA from M. tuberculosis H37Rv, a
118
clinical M. tuberculosis isolate with the Ser531Leu mutation in rpoB and a clinical M.
119
tuberculosis isolate with the Arg548His mutation in rpoB (MTBR548H) were used as
120
templates. The PCR reactions began with a five-minute denaturation at 95 °C, followed by 30
121
cycles of denaturation at 95 °C for one minute, annealing at 58 °C for one minute, extension at
122
72 °C for two minutes, and a final step at 72 °C for two minutes. The PCR fragments were
123
cloned into the pGEM-T easy plasmid (Promega, Wisconsin, USA), followed by excision with
124
EcoR I/Hind III and ligation into pMV261 (Table 1) (20). The constructs were then confirmed
125
by DNA sequencing. To prepare M. tuberculosis and M. smegmatis competent cells, bacteria
126
were incubated in 7H9 broth containing 0.2 M glycine at 37 ⁰C with shaking at 220 rpm until
127
the OD600 reached 0.8~1.0. Subsequently, the bacterial cells were washed three times with
128
ice-cold 10% (w/v) glycerol. Finally, the competent cells were suspended in ice-cold 10%
129
(w/v) glycerol, and the recombinant plasmids pMV261::rpoB_WTc, pMV261::rpoB_S531Lc
130
and pMV261::rpoB_R548Hc were transformed respectively into competent M. tuberculosis
131
H37Rv and M. smegmatis mc2155 cells by electroporation (BTX ECM630 system, Harvard
132
Apparatus, MA, USA) at 2500 V, 1000 Ω and 25 μF.
133 7
134
Drug susceptibility testing. Drug susceptibility testing for mycobacterial clinical isolates was
135
determined by the standard agar proportion method using 1μg/mL of rifampicin as breakpoint
136
(21). The minimum inhibitory concentration (MIC) was determined using standard agar
137
proportion method on 7H11 agar for recombinant M. tuberculosis and broth microdilution
138
method for recombinant M. smegmatis (22). In brief, aliquots of recombinant M. smegmatis
139
(5×103 cells/ml) was inoculated into a 96-well microtitre plate containing 2-fold serial
140
dilutions of rifampicin (from 0.125 to 64 μg/mL) in Müller-Hinton broth with OADC. Then,
141
the bacteria were incubated at 37 ⁰C and 200 rpm for three to five days. Aliquots of
142
recombinant M. tuberculosis and 1:100 dilution control were inoculated on 7H11 agar
143
containing OADC and 2-fold serial dilutions of rifampicin (from 0.125 to 64 μg/mL). The
144
experiments of MIC test were performed three times. The MIC value for each strain was
145
defined as the lowest concentration of rifampicin needed to inhibit bacterial growth.
146 147
Structural modeling. Structural models of M. tuberculosis RpoB bound to rifampicin
148
or DNA were developed by SWISS-MODEL, a fully automated protein structure
149
homology-modelling server, using Protein Data Bank (PDB) accession code 1I6V (Thermus
150
aquaticus RNAP complexed with rifampicin) or 4G7Z (Thermus thermophiles RNAP
151
complexed with DNA) as the respective templates. The M. tuberculosis RpoB Arg548His
152
(using E. coli residue numbering) mutant was generated by Coot (23) using the RpoB-DNA 8
153
modelling structure. All structural models were optimised by energy minimisation using
154
REFMAC5 (24) in the CCP4 program suite (25, 26). Prior to generating structural figures, all
155
models were superimposed by the secondary structure matching (SSM) algorithm of the
156
PDBeFold server (27). The structural figures were produced using PyMOL (DeLano Scientific,
157
http://www.pymol.org).
158
9
159
RESULTS
160
Genetic analysis of rpoB genes in rifampicin-resistant M. tuberculosis isolated from
161
eastern Taiwan. Fifty-one rifampicin-resistant isolates were collected from eastern Taiwan.
162
To determine whether the DNA sequence outside of the RRDR of rpoB that codes for 507-533
163
of RpoB (using E. coli numbering) was associated with rifampicin resistance in M.
164
tuberculosis, a 1681-bp DNA fragment containing RRDR was amplified from each
165
rifampicin-resistant isolate by PCR and analysed by DNA sequencing. After comparison with
166
the rpoB sequence of M. tuberculosis H37Rv, the most commonly mutated sites in RpoB were
167
found to be codons 531 (74.5%) and 526 (21.6%), which are both located in the RRDR (Table
168
3). Of the 51 rifampicin-resistant isolates, 50 were mutated within the RRDR, while only one
169
clinical isolate, named MTBR548H, had a single mutation outside of this region. Although
170
there were three isolates having mutation, at codon 500, 552 and 576 respectively, outside of
171
RRDR, they had second or third mutation in the RRDR (Table 3). Most rifampicin-resistant
172
isolates (72.5%) had one mutated codon in RpoB; however, 23.5% and 3.9% of
173
rifampicin-resistant isolates had two and three mutated RpoB codons, respectively (Table 4).
174 175
Mutation of codon 548 in RpoB results in resistance to rifampicin in recombinant
176
M. tuberculosis and M. smegmatis. In the rifampicin-resistant isolates, one novel rpoB allele
177
that contained a single mutation, at codon 548, outside of the 81-bp RRDR was identified in 10
178
the clinical mutant, MTBR548H (Table 3). To determine whether the mutation of codon 548
179
(Arg548His) in RpoB was involved in bacterial resistance to rifampicin, we constructed
180
recombinant M. tuberculosis H37Rv and M. smegmatis mc2155 strains that carried mutated or
181
wild-type rpoB (Table 1). A bacterium carrying rpoB with the mutated codon Ser531Leu was
182
constructed as a positive control due to an association between this common missense
183
mutation of RpoB and high levels of mycobacterial rifampicin resistance (Table 1). Next, the
184
susceptibility of the recombinant strains to rifampicin was tested. The rifampicin MIC for the
185
recombinant M. smegmatis strain containing the wild-type M. tuberculosis rpoB gene (named
186
CH004) was 2 μg/ml. Meanwhile, the recombinant M. smegmatis strains containing RpoB
187
mutated at codon 531 or 548 (named CH006 and CH005, respectively) showed MIC values of
188
16 μg/mL and 4 μg/mL, respectively, which were elevated 8-fold and 2-fold compared with
189
strains containing either the vector control or wild-type rpoB (Table 5). Additionally, M.
190
tuberculosis H37Rv harbouring RpoB (Arg548His), named YY002, had an MIC of 2~4
191
μg/mL, which was elevated more than 16-fold compared with M. tuberculosis harbouring the
192
wild type rpoB (named YY003) (Table 6). The M. tuberculosis H37Rv harbouring RpoB with
193
mutated codon 531 (named YY004) was used as positive control showing rifampicin
194
resistance (Table 6). Thus, our results indicated that a missense mutation in RpoB at codon
195
548 (Arg548His), which is located outside of the RRDR, slightly increased the resistance of
196
mycobacteria to rifampicin. 11
197 198
Structural models of M. tuberculosis RpoB bound to rifampicin or promoter DNA.
199
To address the structural effect of the Arg548His RpoB mutation on M. tuberculosis
200
rifampicin resistance, structural models of RpoB bound to rifampicin or DNA were generated
201
by a homology-modelling server. In the RpoB-rifampicin complex model, it appeared that
202
residue Arg548 was shifted away from the rifampicin-binding pocket of RpoB and
203
nonessential residues for RpoB binding to rifampicin (Fig. 1). However, in the RpoB-DNA
204
complex model, it appeared that residue Arg548 was close to the rifampicin-resistant cluster II
205
region of RpoB (Fig. 1) (5). In the Arg548His mutant of the RpoB-DNA complex model, the
206
imidazole ring of His548 seemed to provide two hydrogen bonds, between His548 and Ile569
207
as well as between His548 and Ala543 (Fig. 1B), leading to increased rigidity of the
208
rifampicin-resistant cluster II region of RpoB (Fig. 1C). We also found dramatic variations in
209
the side-chain orientation of the rifampicin-resistant cluster II region of RpoB in the
210
rifampicin- and DNA-binding models (Fig. 1C). We speculated that the side chain of His548
211
could provide a more stable hydrogen bond-linking structure than Arg548, thus reducing the
212
flexibility of the rifampicin-resistant cluster II region of RpoB. Thus, the rifampicin-resistant
213
cluster II region of the Arg548His RpoB mutant was unable to interact effectively with
214
rifampicin, resulting in bacterial rifampicin resistance.
215
DISCUSSION 12
216
Although mycobacterial resistance to rifampicin is primarily mediated by mutations within the
217
RRDR, many studies have reported novel mutations outside this region, such as at codons 176
218
(Val176Phe) (10), 381 (Ala381Val) (11), 490 (Gln490His) (12), 500 (Ala500Val), 502
219
(Ile502Val), 505 (Phe505Ser), 538 (Leu538Pro) (13), 146 (Val146Phe) and 572 (Ile572Phe)
220
(14). These mutations were discovered using DNA sequencing of clinical rifampicin-resistant
221
M. tuberculosis isolates; however, most of these findings lacked in vivo confirmation of the
222
relationship between the mutated codon and drug resistance. An exception to this is the work
223
by Siu et al., who constructed recombinant M. smegmatis and M. tuberculosis to show that
224
mutations in codons 146 (Val146Phe) and 572 (Val572Phe) led to rifampicin resistance (14).
225
Compared with western, northern and southern Taiwan, the topography and population
226
composition are different in eastern Taiwan. Therefore, we screened the rpoB gene in 51
227
clinical rifampicin-resistant M. tuberculosis isolates in eastern Taiwan and identified one
228
isolate, MTBR548H, having a mutation in codon 548, which is located outside the RRDR and
229
had not been previously reported, as a cause of bacterial rifampicin resistance. To validate the
230
association of this mutated genotype to the rifampicin-resistant phenotype in MTBR548H, the
231
mutated rpoB of MTBR548H was cloned in multicopy-number plasmid and transformed into
232
wild-type M. tuberculosis and M. smegmatis to produce resistant mutant strains, YY002 and
233
CH005 respectively. The rifampicin susceptibilities of recombinant strains indicated that the
234
Arg548His mutation in RpoB contributed to rifampicin resistance in M. tuberculosis. This 13
235
finding was supported by data obtained from RpoB-DNA structural modelling, which showed
236
that RpoB residue His548 reduced the interaction between RpoB and rifampicin in the
237
RpoB-DNA complex. Our findings could help to improve the detection of mycobacterial
238
rifampicin resistance, and we suggest that position 548 should be included when the RRDR is
239
analysed by DNA sequencing due its close proximity to the RRDR. This can be readily
240
performed without increasing the turnaround time or reaction cost of single-sequencing
241
reactions.
242
Here, the most common mutations involved in rifampicin resistance were missense
243
mutations in codon 531 of rpoB (Table 3). These results were comparable to those of previous
244
studies (6), but the frequency of this mutation (74.5%) was much higher than in earlier reports
245
from other regions of Taiwan, including southern Taiwan (41.5% during 1996-1998), the
246
entire Taiwan (49.4% during 1998-2003) and central and northern Taiwan (68.2% during
247
1999-2011) (6, 15, 16). This difference in frequency could be due to the increasing prevalence
248
of rifampicin-resistant M. tuberculosis clones spreading through eastern Taiwan in recent
249
years (2011-2012).
250
In the recent study in Canada, only 2.9% of clinical rifampicin-resistant isolates (1/35)
251
had double point mutations in rpoB (28). However, we found high ratios of clinical
252
rifampicin-resistant isolates having two or three point mutations in rpoB. (23.5% and 3.9%
253
respectively, Table 4). In our study, most of double or triple point mutations in rpoB were still 14
254
located in RRDR (Table 3). The relationship between the multi-mutations in rpoB and MIC
255
values are not clear and needed further study.
256
The population of aboriginal people in eastern Taiwan is more than other regions in
257
Taiwan. In recent study, 5.43% of drug-resistant TB cases were rifampicin monoresistant in
258
aboriginal people in eastern Taiwan while 2.27% in aboriginal people in southern Taiwan (29).
259
In non-aboriginal people in southern Taiwan from 2010 to 2011 and eastern Taiwan from
260
2005 to 2008 as well as in people in Canada during 2012, no rifampicin-monoresistant clinical
261
isolate was found (30, 31). The novel mutant rpoB (R548H) found in our study might occur
262
due to this special populational and geographical environment. Such mutant strain may spread
263
to other regions in the future if the efforts of control of tuberculosis were not effective.
264
The mutation at codon 531 was associated with high-level resistance to rifampicin in
265
most clinical and recombinant strains (Table 3, 5 and 6) (15, 20). Extrapolation from the
266
crystal structure of the rifampicin-RNAP (RNA polymerase) complex showed that Ser531 in
267
the RpoB rifampicin-binding pocket formed a direct hydrogen bond with the critical hydroxyl
268
of rifampicin, O2. Thus, mutation of codon 531 reduced the interaction between rifampicin
269
and RpoB, resulting in bacterial rifampicin resistance (5). Siu et al. reported that mutation of
270
residues Val146 and Ile572, which are outside the RRDR, resulted in M. tuberculosis
271
resistance to rifampicin. In a model of rifapentine-RRDR (rather than rifampicin-RRDR),
272
residue 572 was localised to the wall of the rifampicin-binding pocket of RpoB, while residue 15
273
146 was located beneath the rifampicin-binding pocket and did not directly interact with the
274
drug. Mutation of residue 572 (Ile572Phe) likely reduced the affinity between that residue and
275
rifampicin, while mutation of residue 146 (Val146Phe) likely affected the folding and packing
276
of the rifampicin-binding pocket (14). In our study, RpoB residue Arg548, which is outside
277
the RRDR, was not located in the rifampicin-binding pocket in the RpoB-rifampicin model.
278
Therefore, residue Arg548 does not directly interact with rifampicin. However, a point
279
mutation (CGC to CAC) in codon 548, which changes the residue from Arg to His, led to
280
reduced flexibility in the RpoB structure (Fig. 1B and 1C). These results indicated that
281
rifampicin could not block the transcriptional activity of RNAP containing a mutated form of
282
RpoB when the RNAP was bound to DNA. Together, our results provide a potential
283
mechanism for the differences in rifampicin resistance between the RpoB-DNA and
284
RpoB-rifampicin models.
285 286
16
287
ACKNOWLEDGMENTS
288
This work was supported partly by grants (contract number NSC 101-2320 -B-320 -002 and
289
MOST 103-2320-B-320-008-MY3) from Ministry of Science and Technology and partly by
290
grants TCIRP99002-04 from Tzu Chi University. We thank the Core Facilities for Protein
291
Structural Analysis in Academia Sinica (Taipei, Taiwan) to assist us in structural modelling
292
and expanding. We also thank Mr. Chi-Hsien Fu and Miss Ying-Huei Chen in Department of
293
Laboratory Medicine Buddhist Tzu Chi General Hospital for their kind help to collect the
294
clinical mycobacterial isolates.
295
17
296
REFERENCES
297
1.
298 299
World Health Organization. 2013. Global tuberculosis report 2013. http://www.who.int/tb/publications/global_report/gtbr13_main_text.pdf.
2.
Centers for Disease Control DoH, R.O.C. (Taiwan). 2012. Taiwan tuberculosis control
300
report 2012
301 302
(http://www.cdc.gov.tw/infectionreportinfo.aspx?treeid=075874dc882a5bfd&nowtre eid=54c73190d38c0d49&tid=3AA3338E6C212AA3).
303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Mitchison DA, Nunn AJ. 1986. Influence of initial drug resistance on the response to short-course chemotherapy of pulmonary tuberculosis. The American review of respiratory disease 133:423-430. Somoskovi A, Parsons LM, Salfinger M. 2001. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respiratory research 2:164-168. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst SA. 2001. Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell 104:901-912. Jou R, Chen HY, Chiang CY, Yu MC, Su IJ. 2005. Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates and identification of 11 novel rpoB alleles in Taiwan. Journal of clinical microbiology 43:1390-1394. Ramirez MV, Cowart KC, Campbell PJ, Morlock GP, Sikes D, Winchell JM, Posey JE. 2010. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis. Journal of clinical microbiology 48:4003-4009. Kumar P, Balooni V, Sharma BK, Kapil V, Sachdeva KS, Singh S. 2014. High degree of multi-drug resistance and hetero-resistance in pulmonary TB patients from Punjab state of India. Tuberculosis 94:73-80. Ohno H, Koga H, Kohno S, Tashiro T, Hara K. 1996. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrobial agents and chemotherapy 40:1053-1056. Heep M, Rieger U, Beck D, Lehn N. 2000. Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy 44:1075-1077. Taniguchi H, Aramaki H, Nikaido Y, Mizuguchi Y, Nakamura M, Koga T, Yoshida S. 1996. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol Lett 144:103-108. Minh NN, Van Bac N, Son NT, Lien VT, Ha CH, Cuong NH, Mai CT, Le TH. 2012. Molecular characteristics of rifampin- and isoniazid-resistant Mycobacterium 18
333 334
tuberculosis strains isolated in Vietnam. Journal of clinical microbiology 50:598-601. 13.
Yue J, Shi W, Xie J, Li Y, Zeng E, Wang H. 2003. Mutations in the rpoB gene of
335
multidrug-resistant Mycobacterium tuberculosis isolates from China. Journal of clinical
336
microbiology 41:2209-2212.
337
14.
Siu GK, Zhang Y, Lau TC, Lau RW, Ho PL, Yew WW, Tsui SK, Cheng VC, Yuen KY, Yam
338
WC. 2011. Mutations outside the rifampicin resistance-determining region associated
339 340
with rifampicin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 66:730-733.
341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
Hwang HY, Chang CY, Chang LL, Chang SF, Chang YH, Chen YJ. 2003. Characterization of rifampicin-resistant Mycobacterium tuberculosis in Taiwan. Journal of medical microbiology 52:239-245. Lin YH, Tai CH, Li CR, Lin CF, Shi ZY. 2013. Resistance profiles and rpoB gene mutations of Mycobacterium tuberculosis isolates in Taiwan. J Microbiol Immunol Infect 46:266-270. Tseng ST, Tai CH, Li CR, Lin CF, Shi ZY. 2013. The mutations of katG and inhA genes of isoniazid-resistant Mycobacterium tuberculosis isolates in Taiwan. J Microbiol Immunol Infect. Nolte FS, Metchock B, Williams T, Diem L, Bressler A, Tenover FC. 1995. Detection of penicillin-resistant Streptococcus pneumoniae with commercially available broth microdilution panels. Journal of clinical microbiology 33:1804-1806. Soo PC, Horng YT, Chang KC, Wang JY, Hsueh PR, Chuang CY, Lu CC, Lai HC. 2009. A simple gold nanoparticle probes assay for identification of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex from clinical specimens. Molecular and cellular probes 23:240-246. Nakata N, Kai M, Makino M. 2012. Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis. Antimicrobial agents and chemotherapy 56:2008-2013. Clinical and Laboratory Standards Institute. 2011. Susceptibility Testing of Mycobacteria, Nocardia and Other Aerobic Actinomycetes: Approved Standard-second edition M24-A2. CLSI, Wayne, PA, USA. Wiegand I, Hilpert K, Hancock RE. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163-175. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta crystallographica. Section D, Biological crystallography 60:2126-2132. Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta crystallographica. Section D, Biological crystallography 67:355-367. 19
371
25.
Collaborative Computational Project N. 1994. The CCP4 suite: programs for protein
372
crystallography. Acta crystallographica. Section D, Biological crystallography
373
50:760-763.
374
26.
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM,
375
Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS,
376
Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS. 2011. Overview of the CCP4
377 378
suite and current developments. Acta crystallographica. Section D, Biological crystallography 67:235-242.
379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398
27.
28.
29.
30. 31.
32. 33.
Krissinel E, Henrick K. 2004. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta crystallographica. Section D, Biological crystallography 60:2256-2268. Jamieson FB, Guthrie JL, Neemuchwala A, Lastovetska O, Melano RG, Mehaffy C. 2014. Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis. Journal of clinical microbiology 52:2157-2162. Chen YY, Chang JR, Huang WF, Kuo SC, Yeh JJ, Lee JJ, Jang CS, Sun JR, Chiueh TS, Su IJ, Dou HY. 2014. Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006-2011. The Journal of infection 68:332-337. Public Health Agency of Canada. 2012. Tuberculosis: Drug resistance in Canada-2012. http://www.phac-aspc.gc.ca/tbpc-latb/pubs/tb-dr2012/index-eng.php#results_1. Chen YY, Tseng FC, Chang JR, Kuo SC, Lee JJ, Yeh JJ, Chiueh TS, Sun JR, Su IJ, Dou HY. 2014. Distinct modes of transmission of tuberculosis in aboriginal and non-aboriginal populations in taiwan. PloS one 9:e112633. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. Journal of molecular biology 166:557-580. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, Bansal GP, Young JF, Lee MH, Hatfull GF, et al. 1991. New use of BCG for recombinant vaccines. Nature 351:456-460.
399
20
400
Figure legend
401
Figure 1. Structural models of M. tuberculosis RpoB that bound with rifampicin or promoter
402
DNA. (A) Superimposition of the models of M. tuberculosis RpoB that bound with rifampicin
403
(protein in gray cartoon model) or promoter DNA (protein in light-orange cartoon model). The
404
rifampicin is shown in stick model (carbon atoms in dark-green). The DNA is shown in sphere
405
model (carbon atoms in light-green and yellow-orange). The carbon atoms of key residues for
406
the RpoB-rifampicin complex, RpoB-DNA complex, and RpoB mutant-DNA complex are
407
shown in light-green, cyan, magenta line models, respectively. Oxygen atoms are shown in red,
408
nitrogen atoms are in blue, and phosphorus atoms are in orange. (B) and (C) Close-up view of
409
the Arg548 located loop and the rifampicin-resistant cluster II region of RpoB. The potential
410
hydrogen bonds are indicated by black dashed lines. The color indication of residues numbers
411
are the same as the color painting of line models. The residues number are labeled according
412
to the E. coli RpoB protein sequence. Values in the parentheses are for M. tuberculosis RpoB
413
protein sequence. (D) Protein sequence alignment of the Arg548 located loop and the
414
rifampicin-resistant cluster II loop of four prokaryotic RpoB. The rifampicin-resistant cluster
415
II region of RpoB is marked with black bar. The Arg548His mutant site and the residues
416
interacting with His548 by potential hydrogen-bond are indicated by black asterisk and dots,
417
respectively.
418
21
419
Table 1. Strains and plasmids used in this study. Strain or plasmid
descriptions
Reference or source
Clinical isolates resistant to rifampicin not to isoniazid
This study
E. coli DH5α
supE44∆lacU169 (f80 lacZ∆M15)hsdR1 recA1 endA1 gyrA96 thi-1 relA1
(32)
M. smegmatis mc2155
Wild type
ATCC700084
Strains M. tuberculosis MTBR548H
2
CH003
M. smegmatis mc 155 / pMV261 vector only, Kmr
This study
CH004
M. smegmatis mc2155 / pMV261::rpoB_WTc , Kmr
This study
CH005
M. smegmatis mc2155/ pMV261::rpoB_R548Hc, Kmr
This study
CH006
M. smegmatis mc2155/ pMV261::rpoB_S531Lc, Kmr
This study
Wild type
ATCC27294
YY001
M. tuberculosis H37Rv / pMV261 vector only, Kmr
This study
YY002
M. tuberculosis H37Rv / pMV261::rpoB_R548Hc, Kmr
This study
YY003
M. tuberculosis H37Rv / pMV261::rpoB_WTc, Kmr
This study
YY004
M. tuberculosis H37Rv / pMV261::rpoB_S531Lc, Kmr
This study
pGEM-T easy
Cloning vector, Ampr
Promega, Wisconsin, USA
pMV261
Mycobacterium species/E. coli shuttle vector, Kmr pMV261 containing wild-type rpoB from M. tuberculosis H37Rv, Kmr
(33)
pMV261::rpoB_R548Hc
pMV261 containing mutated rpoB from rifampicin-resistant M. tuberculosis, codon 548 of RpoB was changed to histidine Kmr
This study
pMV261::rpoB_S531Lc
pMV261 containing mutated rpoB from rifampicin-resistant M. tuberculosis, codon 531 of RpoB was changed to lysine, Kmr
This study
M. tuberculosis H37Rv
plasmids
pMV261::rpoB_WTc
420 22
This study
421
Table 2 primers designed in this study Primer
Sequence (5’-3’)
Amplicon size
rpoB-FP
CCCGCCAGAGCAAAACAGC
rpoB-RP
TACTCCACCTCGCCCGCC
rpoB-seq-F
AATATCTGGTCCGCTTGCAC
rpoB-seq-R
ACGAGGGCACGTACTCCA
rpoB-full-cF
GCGAATTCGGAAGGAAAAATGGCAGATTCCCGCCAG
rpoB-full-cR
GCAAGCTTTTACGCAAGATCCTCGACAC
1681 bp
422
23
3545 bp
423
Table3. Mutation in rpoB gene in 51 rifampicin-resistant M. tuberculosis Mutated codon Ala500Gly
AGCAAC
1
2.0
a
ATGTTG
1
2.0
a
GACGTC
3
5.9
a
CACTAC
1
2.0
a
His526Asn
Arg548His Pro552Ser Pro567Ser
424 425 426 427 428 429 430
Frequency (%)
1
Asp516Val
Leu533Pro
b
GCCGGC
Met515Leu
Ser531Leu
Number of strains
a
Ser512Asn
His526Tyr
Nucleic acid change
2.0
CACAAC
10
19.6
a
TCGTTG
38
74.5
a
CTGCCG
10
19.6
1
2.0
CGCCAC
e
CCGTCG CCTTCT
1
c
1
2.0
d
2.0
Note: a, The mutations located within the 81-bp RRDR of rpoB gene. There were 11 isolates having two mutated codons both in the RRDR b, The isolate had three mutated codons in RpoB: Ala500Gly, Met515Leu and Asp516Val. c, The isolate had two mutated codons in RpoB: Ser531Leu and Pro552Ser. d, The isolate had three mutated codons in RpoB: Ser531Leu, Leu533Pro and Pro567Ser. e, The novel allele was identified in this study.
431
24
432
Table 4. The frequency of single or multi mutations in RpoB in 51
433
rifampicin-resistant M. tuberculosis Mutation in RpoB
No. of strains
Frequency
One codon
37
72.5%
Two codons
12
23.5%
Three codons
2
3.9%
Four codons
0
0%
434
25
435 436
Table 5. Rifampicin susceptibility of the recombinant M. smegmatis strains with wild type RpoB or mutated RpoB M. smegmatis strains
Codon no. and amino acid
Rifampicinb MIC(μg/mL)
CH003
pMV261a
2
CH004
wild type rpoB
2
CH005
Arg548His
4
CH006
Ser531Leu
16
437
a, Vector control
438
b, Each MIC test was performed three times.
439
26
440 441
Table 6. Rifampicin susceptibility of the recombinant M. tuberculosis strains with wild type RpoB or mutated RpoB using agar proportion method . 10-4 dilutionb
10-2 dilutionb M. tuberculosis strains
Codon no. and amino acid
Rifampicinc MIC(μg/mL)
Sensitive /Resistantd
Rifampicinc MIC(μg/mL)
Sensitive /Resistantd
YY001
pMV261 a
